trpm7 sirna  (Santa Cruz Biotechnology)

Journal: Advanced Science

Article Title: Thermogenic Adipocytes Promote M2 Macrophage Polarization through CNNM4‐Mediated Mg Secretion

doi: 10.1002/advs.202401140

Figure Lengend Snippet: CNNM4 mediates Mg release from thermogenic adipocytes. A) Representative staining of TMRM, MitoTracker, and Mag‐Fluo‐4 in beige adipocytes treated with indicated compounds (CL316243 2 µg mL −1 , H89 10 × 10 −6 m , FCCP 5 × 10 −6 m , CsA 100 × 10 −9 m ). Scale bar, 20 µm; B) Mg transmembrane transporter expression in adipocytes from mice under cold exposure or RT through single cell sequencing from literature; C) fold change of the Mg transporters gene expression ( Mrs2, Cnnm4, Cnnm3, Slc41a1, Slc41a2, Magt1, Trpm6 , and Trpm7 ) in scWAT, SVF, or adipocytes under cold exposure or CL316243 stimulation; D) UCP1 and CNNM4 protein expression in scWAT from mice under room temperature (25 °C) or cold exposure (4 °C); E–G) UCP1 and CNNM4 protein expression in scWAT from mice with 6‐OHDA injection, surgical transection of sympathetic neuron fibers, or local injection with L748337 under cold exposure; H) UCP1 and CNNM4 protein expression and CREB phosphorylation in primary beige adipocytes treated with CL316243 (2 µg mL −1 ) together with L748337 (4 × 10 −6 m ) or H89 (10 × 10 −6 m ); I) ChIP‐qPCR analysis of CREB binding to CNNM4 promoter in primary beige adipocytes treated with CL316243. Result was normalized to input control values and represented as fold enrichment relative to the anti‐rabbit IgG control ( n = 3); J) Mg levels in scWAT‐derived interstitial fluid from CNNM4 Adipoq‐cKO or WT mice ( n = 6); K) Mg levels in scWAT‐derived interstitial fluid from C57BL/6 mice with local injection of Adv‐GFP or Adv‐CNNM4 ( n = 6). Data were expressed as means ± SEM. (B), (C), (J), and (K) were calculated by unpaired two‐tailed Student's t ‐test; (I) were calculated by two‐way ANOVA followed with Bonferroni's multiple comparison test.

Article Snippet: For TRPM7 silencing, BMDMs were transfected with 10 × 10 −9 m siRNA targeting mice TRPM7 (SR423257, OriGene, USA) following the manufacturer's instructions using siTran1.0 (Ori‐Gene, USA) as the agent.

Techniques: Staining, Expressing, Sequencing, Gene Expression, Injection, Phospho-proteomics, ChIP-qPCR, Binding Assay, Control, Derivative Assay, Two Tailed Test, Comparison

Journal: Advanced Science

Article Title: Thermogenic Adipocytes Promote M2 Macrophage Polarization through CNNM4‐Mediated Mg Secretion

doi: 10.1002/advs.202401140

Figure Lengend Snippet: CNNM4 expression is associated with obesity. A) Representative flow cytometric plots and quantification demonstrate the numbers of M2 macrophages (CD206 + /CD11c − ) in scWAT of mice fed with HFD or regular diet (RD) for 12 weeks ( n = 3); B) representative M1 and M2 macrophage marker gene expression in scWAT from lean and obese human individuals (16 vs 16); C) volcano plot of ions altered in scWAT‐derived interstitial fluid from HFD or RD‐fed mice ( n = 4); D) Mg levels in scWAT‐derived interstitial fluid from HFD or RD‐fed mice ( n = 6); E) representative Mg transporters ( Mrs2, Cnnm4, Cnnm3, Slc41a1, Slc41a2, Magt1, Trpm6 , and Trpm7 ) gene expression in scWAT from HFD or RD‐fed mice ( n = 6) or F) lean and obese human individuals (16 vs 16); G) representative CNNM4 and UCP1 protein expression in scWAT from lean and obese human individuals; H) the correlation between BMI and blood glucose, I) representative M1 and M2 macrophage marker gene expression ( CD68, RANTES, MCP1, TNFα, CD301, MRC1, ARG1 , and IL10 ) with CNNM4 expression in lean and obese human individuals ( n = 32). Data were expressed as means ± SEM. (A), (B), and (D)–(F) were calculated by unpaired two‐tailed Student's t ‐test or (H) and (I) were analyzed by Pearson correlation analysis.

Article Snippet: For TRPM7 silencing, BMDMs were transfected with 10 × 10 −9 m siRNA targeting mice TRPM7 (SR423257, OriGene, USA) following the manufacturer's instructions using siTran1.0 (Ori‐Gene, USA) as the agent.

Techniques: Expressing, Marker, Gene Expression, Derivative Assay, Two Tailed Test

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Thermogenic Adipocytes Promote M2 Macrophage Polarization through CNNM4-Mediated Mg Secretion.

doi: 10.1002/advs.202401140

Figure Lengend Snippet: Figure 3. CNNM4 mediates Mg release from thermogenic adipocytes. A) Representative staining of TMRM, MitoTracker, and Mag-Fluo-4 in beige adipocytes treated with indicated compounds (CL316243 2 μg mL−1, H89 10 × 10−6 m, FCCP 5 × 10−6 m, CsA 100 × 10−9 m). Scale bar, 20 μm; B) Mg transmembrane transporter expression in adipocytes from mice under cold exposure or RT through single cell sequencing from literature; C) fold change of the Mg transporters gene expression (Mrs2, Cnnm4, Cnnm3, Slc41a1, Slc41a2, Magt1, Trpm6, and Trpm7) in scWAT, SVF, or adipocytes under cold exposure or CL316243 stimulation; D) UCP1 and CNNM4 protein expression in scWAT from mice under room temperature (25 °C) or cold exposure (4 °C); E–G) UCP1 and CNNM4 protein expression in scWAT from mice with 6-OHDA injection, surgical transection of sympathetic neuron fibers, or local injection with L748337 under cold exposure; H) UCP1 and CNNM4 protein expression and CREB phosphorylation in primary beige adipocytes treated with CL316243 (2 μg mL−1) together with L748337 (4 × 10−6 m) or H89 (10 × 10−6 m); I) ChIP-qPCR analysis of CREB binding to CNNM4 promoter in primary beige adipocytes treated with CL316243. Result was normalized to input control values and represented as fold enrichment relative to the anti-rabbit IgG control (n = 3); J) Mg levels in scWAT-derived interstitial fluid from CNNM4Adipoq-cKO or WT mice (n = 6); K) Mg levels in scWAT-derived interstitial fluid from C57BL/6 mice with local injection of Adv-GFP or Adv-CNNM4 (n = 6). Data were expressed as means ± SEM. (B), (C), (J), and (K) were calculated by unpaired two-tailed Student’s t-test; (I) were calculated by two-way ANOVA followed with Bonferroni’s multiple comparison test.

Article Snippet: TRPM7 Inhibition and Blockage: For TRPM7 silencing, BMDMs were transfected with 10 × 10−9 m siRNA targeting mice TRPM7 (SR423257, OriGene, USA) following the manufacturer’s instructions using siTran1.0 (Ori-Gene, USA) as the agent.

Techniques: Staining, Expressing, Sequencing, Gene Expression, Injection, Phospho-proteomics, ChIP-qPCR, Binding Assay, Control, Derivative Assay, Two Tailed Test, Comparison

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Thermogenic Adipocytes Promote M2 Macrophage Polarization through CNNM4-Mediated Mg Secretion.

doi: 10.1002/advs.202401140

Figure Lengend Snippet: Figure 5. CNNM4 expression is associated with obesity. A) Representative flow cytometric plots and quantification demonstrate the numbers of M2 macrophages (CD206+/CD11c−) in scWAT of mice fed with HFD or regular diet (RD) for 12 weeks (n = 3); B) representative M1 and M2 macrophage marker gene expression in scWAT from lean and obese human individuals (16 vs 16); C) volcano plot of ions altered in scWAT-derived interstitial fluid from HFD or RD-fed mice (n = 4); D) Mg levels in scWAT-derived interstitial fluid from HFD or RD-fed mice (n = 6); E) representative Mg transporters (Mrs2, Cnnm4, Cnnm3, Slc41a1, Slc41a2, Magt1, Trpm6, and Trpm7) gene expression in scWAT from HFD or RD-fed mice (n = 6) or F) lean and obese human individuals (16 vs 16); G) representative CNNM4 and UCP1 protein expression in scWAT from lean and obese human individuals; H) the correlation between BMI and blood glucose, I) representative M1 and M2 macrophage marker gene expression (CD68, RANTES, MCP1, TNF𝛼, CD301, MRC1, ARG1, and IL10) with CNNM4 expression in lean and obese human individuals (n = 32). Data were expressed as means ± SEM. (A), (B), and (D)–(F) were calculated by unpaired two-tailed Student’s t-test or (H) and (I) were analyzed by Pearson correlation analysis.

Article Snippet: TRPM7 Inhibition and Blockage: For TRPM7 silencing, BMDMs were transfected with 10 × 10−9 m siRNA targeting mice TRPM7 (SR423257, OriGene, USA) following the manufacturer’s instructions using siTran1.0 (Ori-Gene, USA) as the agent.

Techniques: Expressing, Marker, Gene Expression, Derivative Assay, Two Tailed Test

Journal: Cells

Article Title: Expression Profiling Identified TRPM7 and HER2 as Potential Targets for the Combined Treatment of Cancer Cells

doi: 10.3390/cells13211801

Figure Lengend Snippet: Assessment of human leukemia HAP1 cells. ( A ) Left panel: Whole-cell currents measured at −80 and +80 mV over time in WT (WT) and two TRPM7 KO (KO-01 and KO-04) HAP1 cell lines. Middle panel: Representative current−voltage (I−V) relationships obtained at 300 s in measurements illustrated on the Left panel. Right panel: Bar graphs of current amplitudes at +80 mV (300 s) illustrated on the Left panel. Data are mean ± SD; n , the number of cells examined. ** p ≤ 0.01 (one-way ANOVA). ( B ) Left panel: Whole-cell currents measured at −80 and +80 mV over time in WT HAP1 cells in the absence (Control) and the presence of 10 or 30 µM NS8593. Middle panel: Representative I−V relationships obtained at 300 s in measurements illustrated on the Left panel. Right panel: Bar graphs of current amplitudes at +80 mV (300 s) illustrated on the Left panel. Data are mean ± SD; n , the number of cells examined. *** p ≤ 0.001 (one-way ANOVA). ( C , D ) Proliferation rate of WT and TRPM7 KO-01 ( C ) and KO-04 ( D ) HAP1 cells. The cells were cultured for 3 days in the regular cell culture medium. The initial cell density (Day 0) was accounted as 100%. Data are mean ± SD of n = 3 independent experiments. *** p ≤ 0.001; ** p ≤ 0.01 ( t -test). ( E ) Viability of WT HAP1 cells maintained in regular cell culture medium (Control) or medium with an additional 10 mM MgCl 2 (Mg suppl) containing different concentrations of NS8593 for 72 h. Cell densities in the absence of NS8593 were accounted as 100%. Data are mean ± SD of n = 3 independent experiments. *** p ≤ 0.001; ** p ≤ 0.01; ns—not significantly different ( t -test).

Article Snippet: The next day, the cells were transiently transfected with 20 nM FlexiTube siRNAs silencing TRPM7 : Hs_ TRPM7 _7 FlexiTube siRNA (siRNA #7), Hs_ TRPM7 _8 FlexiTube siRNA (siRNA #8), and AllStars Negative Control siRNA (all from QIAGEN, Hilden, Germany) using Lipofectamine 2000 reagent (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Control, Cell Culture

Journal: Cells

Article Title: Expression Profiling Identified TRPM7 and HER2 as Potential Targets for the Combined Treatment of Cancer Cells

doi: 10.3390/cells13211801

Figure Lengend Snippet: Genome-wide transcriptome profiling of HAP1 cells. ( A ) Venn diagrams for transcripts showing ≥1.5-fold up-regulation (Left panel) and down-regulation (Right panel) in TRPM7 KO-01 (KO-01), TRPM7 KO-04 (KO-04), and NS8593-treated WT (NS8593) HAP1 cells as compared to untreated WT HAP1 cells. ( B – D ) Relative expression levels of HER2 (ERBB2) ( B ) and HER3 (ERBB3) ( C ) and ALPK1 ( D ), assessed by qRT-PCR approach in WT (WT), TRPM7 KO-01 (KO-01), TRPM7 KO-04 (KO-04), and NS8593-treated WT (WT+NS8593) HAP1 cells with HPRT as a reference transcript. Data are mean ± SD of n = 3 independent experiments. *** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05; ns—not significantly different (one-way ANOVA).

Article Snippet: The next day, the cells were transiently transfected with 20 nM FlexiTube siRNAs silencing TRPM7 : Hs_ TRPM7 _7 FlexiTube siRNA (siRNA #7), Hs_ TRPM7 _8 FlexiTube siRNA (siRNA #8), and AllStars Negative Control siRNA (all from QIAGEN, Hilden, Germany) using Lipofectamine 2000 reagent (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Genome Wide, Expressing, Quantitative RT-PCR

Journal: Cells

Article Title: Expression Profiling Identified TRPM7 and HER2 as Potential Targets for the Combined Treatment of Cancer Cells

doi: 10.3390/cells13211801

Figure Lengend Snippet: The impact of TRPM7 on HER2 expression levels in HAP1 cells. ( A ) HER2 expression in WT (WT), TRPM7 KO-04 (KO-04), TRPM7 KO-01 (KO-01), and NS8593-treated WT (WT+NS8593) HAP1 cells. ( B – D ) Effects of 10 µM ZnCl 2 (Zn) and 0.5 µM zinc pyrithione (ZP) on HER2 expression in WT (WT) and TRPM7 KO-04 (KO) HAP1 cells. ( E ) HER2 expression in WT HAP cells treated by 20 µM NS8593 for 24, 48, and 72 h. In ( C – E ), equal DMSO volumes (DMSO) were used instead of ZP or NS8593. Upper panels: Representative western blots are shown. Equal volumes of cell lysates were assessed using anti-HER2 and anti-β-actin antibodies. Lower panels: Bar graphs showing normalized HER2 expression levels in experiments from the Upper panel. The ratio of HER2 and anti-β-actin signals in untreated WT HAP1 cells was accounted as 100%. The results shown in the bar graphs are mean ± SD of n = 3 independent experiments. In ( A – D ), *** p ≤ 0.001; ** p ≤ 0.01; ns—not significantly different (one-way ANOVA). In ( E ), ** p ≤ 0.01; * p ≤ 0.05; ns—not significantly different ( t -test).

Article Snippet: The next day, the cells were transiently transfected with 20 nM FlexiTube siRNAs silencing TRPM7 : Hs_ TRPM7 _7 FlexiTube siRNA (siRNA #7), Hs_ TRPM7 _8 FlexiTube siRNA (siRNA #8), and AllStars Negative Control siRNA (all from QIAGEN, Hilden, Germany) using Lipofectamine 2000 reagent (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Western Blot

Journal: Cells

Article Title: Expression Profiling Identified TRPM7 and HER2 as Potential Targets for the Combined Treatment of Cancer Cells

doi: 10.3390/cells13211801

Figure Lengend Snippet: The regulatory effect of TRPM7 on HER2 expression in embryonic trophoblast stem (TS) cells and HEK293 cells. ( A , B ) Assessment of HER2 expression in WT (WT) and TRPM7 KO (KO) TS cells. Upper panels: Representative western blots obtained with the cells cultured in the absence or presence of 10 µM ZnCl 2 (Zn) and 0.5 µM zinc pyrithione (ZP) are shown. Equal volumes of cell lysates were assessed using anti-HER2 and anti-β-actin antibodies. Equal volumes of DMSO (DMSO) were used instead of ZP as an additional control. Lower panels: Bar graphs showing normalized HER2 expression levels in experiments in the Upper panels. The ratio of HER2 to anti-β-actin signals in WT TS cells was accounted as 100%. ( C – E ) Assessment of HER2 expression in WT (WT) and TRPM7 KO (KO) HEK293 cells in the absence or presence of 10 µM ZnCl 2 (Zn) and 0.5 µM zinc pyrithione (ZP). The experiments were performed and analyzed analogously to ( A , B ). ( F ) Analysis of HER2 expression in untransfected WT (Untransfected) HEK293 cells, WT HEK293 cells transfected by TRPM7 -specific siRNA #7 (siRNA #7), TRPM7 -specific siRNA #8 (siRNA # 8 ) and AllStars Negative Control siRNA (Control siRNA), and TRPM7 KO (KO) HEK293 cells. Experiments were performed and analyzed analogously to ( A , B ) except that the ratio of HER2 to anti-β-actin signal in untransfected WT HEK293 cells was accounted as 100%. The results in the bar graphs are mean ± SD of n = 3 independent experiments. *** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05; ns—not significantly different (one-way ANOVA).

Article Snippet: The next day, the cells were transiently transfected with 20 nM FlexiTube siRNAs silencing TRPM7 : Hs_ TRPM7 _7 FlexiTube siRNA (siRNA #7), Hs_ TRPM7 _8 FlexiTube siRNA (siRNA #8), and AllStars Negative Control siRNA (all from QIAGEN, Hilden, Germany) using Lipofectamine 2000 reagent (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Western Blot, Cell Culture, Control, Transfection, Negative Control

Journal: Cells

Article Title: Expression Profiling Identified TRPM7 and HER2 as Potential Targets for the Combined Treatment of Cancer Cells

doi: 10.3390/cells13211801

Figure Lengend Snippet: Assessment of breast cancer SKBR3, MCF-7, and MDA-MB-231 cells. ( A ) Analysis of HER2 expression in SKBR3, MCF-7, and MDA-MB-231 cells. Upper panel: Representative western blot obtained with SKBR3, MCF-7, and MDA-MB-231 cells. Equal volumes of cell lysates were assessed using anti-HER2 and anti-β-actin antibodies. Lower panel: Bar graph showing normalized HER2 expression levels in experiments from the Upper panel. The ratio of HER2 to anti-β-actin signal in SKBR3 cells was accounted as 100%. ( B ) Assessment of HER2 expression in untransfected (Untransfected) SKBR3 cells and SKBR3 cells transfected by TRPM7 -specific siRNA #7 (siRNA #7), siRNA #8 (siRNA #8), and AllStars Negative Control siRNA (Control siRNA). Experiments were performed and analyzed analogously to ( A ) except that the ratio of HER2 to anti-β-actin signal in untransfected SKBR3 cells was accounted as 100%. The results in the bar graphs in ( A , B ) are mean ± SD of n = 3 independent experiments. ** p ≤ 0.01; * p ≤ 0.05; ns—not significantly different (one-way ANOVA). ( C ) Comparison of endogenous TRPM7 currents measured in SKBR3 and MDA-MB-231 cells. Left panel: Whole-cell currents measured at −80 and +80 mV over time in SKBR3 and MDA-MB-231 cells. Middle panel: Representative current−voltage (I−V) relationships obtained at 300 s in measurements shown in the Left panel. Right panel: Bar graph of current amplitudes at +80 mV (300 s) illustrated on the Left panel. Data are mean ± SD; n , the number of cells examined. Ns—not significantly different ( t -test). ( D ) Viability of SKBR3 and MDA-MB-231 cells exposed to different concentrations of NS8593 for 72 h. Cell densities in the absence of NS8593 were accounted as 100%. Data are mean ± SD of n = 3 independent experiments. *** p ≤ 0.001; ns—not significantly different ( t -test). ( E ) HER2 expression in SKBR3 cells treated by 30 µM NS8593 (NS8593) or equal volumes of DMSO (DMSO) for 72 h. Upper panel: Representative western blots are shown. Equal volumes of cell lysates were assessed using anti-HER2 and anti-β-actin antibodies. Lower panel: Bar graphs showing normalized HER2 expression level in experiments from the Upper panel. The results in the bar graphs are mean ± SD of n = 3 independent experiments. ** p ≤ 0.01 ( t -test).

Article Snippet: The next day, the cells were transiently transfected with 20 nM FlexiTube siRNAs silencing TRPM7 : Hs_ TRPM7 _7 FlexiTube siRNA (siRNA #7), Hs_ TRPM7 _8 FlexiTube siRNA (siRNA #8), and AllStars Negative Control siRNA (all from QIAGEN, Hilden, Germany) using Lipofectamine 2000 reagent (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Western Blot, Transfection, Negative Control, Control, Comparison